Multielectrode array and system for recording and analyzing data or for stimulating tissue

ABSTRACT

Disclosed is a multielectrode array suitable for many biological and analytical functions including manipulation of data from neurons, neural prosthetics and biosensing, together with ancillary devices and software for manipulation of data obtained therefrom.

FIELD OF DISCLOSURE

The present disclosure relates in general to the fields of (1) recordingin bodily tissues or other substances for the presence of variousphenomena such as, for example, action potentials in neurons or pH inliquids, (2) stimulating bodily tissues with appropriate electricalvoltage in the range of millivolts for prosthetics and otherapplications, and (3) recording and analyzing data received from bodilytissues.

BACKGROUND

The central nervous system (CNS) and peripheral nervous system (PNS)relate information by means of neurons which transmit electricalactivation by means of an action potential through release ofneurotransmitters at a chemical synapse or by means of gap junctions inwhich charged ions flow directly between cells. The patterns ofactivations of neurons are the language of the CNS and PNS.Electrophysiology is the study of bioelectrical activation of neuronsand includes the use of instruments such as the present invention inwhich recordings are made of neural activation. Recordings of neuralactivation, as well as other investigative tools, have created knowledgeabout the workings of the CNS and PNS, although there is obviously muchyet to discover.

There has been increasing interest in simultaneous recording of thespiking activity of multiple neurons in the last decade for numerousreasons. One reason is to advance big picture theories of how neuronswork together to allow sensory perception, motor activity and otherneural activities. Fundamental aspects of neural coding, such assynchronous firing (Singer, 1997), which are not discernable from singlecell recordings, have been detected by simultaneous neural recordings.Other hypothesized sensory codes, such as order of firing (Thorpe et al.2001) also require simultaneous, multi-unit recordings. Multielectrodearrays for recording action potentials of neurons (spikes) are alsoequally adaptable as means for stimulating sensory (e.g., retina),brain, or other neural tissue.

Multielectrode arrays have universal application within the nervoussystem, and the retina has been a particularly prominent locus for thedevelopment of multi-unit recording arrays. Compared to “brain slice”preparations that are in a depressed state without most of their input,the retina can be removed without cutting the processes of any cellsexcept the axons of the ganglion cells several millimeters from theirsomas. Since the input to the retina is light, which can be supplied andcontrolled just as well in the dish as in situ, the retina can beoperated in vitro in a nearly normal state of responsiveness for manyhours. A technical advantage for array recording is that in retina allthe spiking ganglion cells are located in a single, accessible layerclose to the surface of the tissue.

There are two basic styles of multielectrode arrays developed for retinarecording with respect to whether the ganglion cell layer to be recordedis on the bottom, or top. One of the bottom recording types is thatdeveloped by Meister, Baylor and colleagues (1994), and consists ofelectrodes mounted on the bottom of a chamber in which the retina isplaced ganglion cell side down, and stimulated with light from above.Bottom-of-chamber configurations have also been reported by Grumet etal. (2000) and Heuschkel et al. (2002), and exist in commercial versionsfor brain slice recordings, with typical inter-electrode spacing andconstruction much different than that of the present invention.Advantages of the dish bottom arrays are that they are rugged, andrecordings are obtained merely by placing the appropriate part of thetissue onto the active part of the array. However, most commerciallyavailable recording arrays are only marginally suitable for addressingcoding issues such as synchronous firing in neural tissue, because fewof the neurons, whose inter-soma spacing can be as low as 20micrometers, will be recorded with electrodes much further apart. Thegreater density of electrodes in a given area of tissue is a factor indetermining whether recording of data gathers the most importantcharacteristics of that tissue. Likewise, greater density of electrodesfor stimulating neurons is more likely to approach the kind of densityand connectivity in all kinds of neural tissue where, for example,cortical neuron somas can be 20 micrometers apart while connected tomore than 5,000 other neurons.

For in vitro experiments, there are also disadvantages to thedish-bottom array approach besides the typical electrode spacingreferred to above. The first is that in order to change the locationwhere the array records, the whole piece of tissue must be physicallymoved. A second problem somewhat particular to retina is thatdish-bottom arrays almost always use the isolated retina preparationthat is much less robust than the isolated eyecup preparation in whichthe retina remains attached to the pigment epithelium. Moreover, forganglion cell recordings, the isolated retina preparation is generallyless healthy mounted ganglion cell side down, than up, becausesuperfusion of the ganglion cell side of the tissue promotes the longterm health of the spiking cells.

As a recording instrument, the present invention in one embodiment hasdemonstrated a better than 50% yield that any given electrode will haveat least one usable recording, with some electrodes yielding 2 or 3usable ganglion cell recordings, so that overall, nearly as manyganglion cells can be recorded as array elements. Retinal recordings arestable for 4-6 hours or more, and different regions of the retina caneasily be investigated by moving the array to a new retinal position.The invention also has the advantage of being usable for long periods oftime, and is easily fabricated by hand using routine technology likelyto be found in any electrophysiology laboratory. The invention isuniquely suited to over-sampling a given area of the neural tissue sothat there is a high probability of recording simultaneously from manyof the neurons in a given area of neural tissue. Likewise, theinvention's small distances between electrodes allow simultaneousstimulation of many of the neurons in a given area of neural tissue.

Multi-array configurations that record from the retina ganglion cellside up have also been developed by Normann and coworkers (1996) andSandison et al. (2002), among others. Recording from above permits easyand rapid movement of the array to an optimum location in the tissuebased on the results obtained. However, in the retina, problems withsome of the extant superior-approaching arrays include inter-elementspacing that is too large, or inability to use the intact retina-pigmentepithelium mounting because the array is not transparent, forcing visualstimulation to come from the side of the tissue opposite from theelectrode. In retina, a relatively transparent multi-electrode array,one of the embodiments described herein, allows recording in either theeyecup or isolated retina preparation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a diagram of a portion of one layer of the multi-electroderecording array. The mica or acetate substrate is glued to theelectrical connector with epoxy. Individually made carbon fiberelectrodes are laid in each layer by stabilizing the electrode tips in afine mesh mounted below the recording end of the substrate (not shown),which is removed after the assembly is complete. The insulated carbonfibers are bonded to fine copper wires, which in turn are soldered tothe electrical connector. Polyurethane insulates the individual carbonfibers, and bonds the assembly together.

FIG. 1B is a diagram of the side view of the multielectrode recordingarray showing how layers are offset with respect to each other, so thatwhen advanced at a 45 degree angle, all electrode tips are in the samehorizontal plane. This permits visualization of the underlying tissuethrough the electrode tips to the substrate, and through the substratefarther from the tips.

FIG. 2A is a copy of a photomicrograph of the tip region of themultielectrode recording array's single insulated and silver-platedcarbon microelectrode. The shiny area is the exposed, plated region. Thescale is 100 microns.

FIG. 2B is a copy of a photomicrograph of a two layer, ten electrodemultielectrode recording array placed over a graticule whose large,labeled divisions are 100 microns apart. The view is from above, throughthe microscope objective, which can also be the stimulus path (althoughstimulation can come from below if an isolated retina, rather thaneyecup is used.) The graticule is clear visible beyond the substrate forapproximately 50 microns. The graticule scale is also visible throughthe substrate at the upper left, although the acetate substraterefraction shifts the image. The refractive shift can be minimized byusing thinner mica rather than acetate as the substrate. The scale is100 microns.

FIG. 3 is a schematic diagram of the electronic amplifier used for eachchannel, with the gain versus frequency plot below. Resistances are inmegaohms (M), capacitors in picofarads (p), and frequency is in logunits (log scale). All operational amplifiers are T1081 equivalent(either T1082 dual or T1084 quad versions are actually used).

FIG. 4 contains traces from simultaneous recordings from the tenelectrode multi-array shown in FIG. 2A and FIG. 2B. Channels 4-8 haveeasily discernable spikes even at this low resolution scale. Asignal-to-noise index was measured as the ratio of the peak to peakspike height to the peak to peak noise 2 ms away for 100 spikes for eachchannel. These values for channels 0-9 are, respectively: 4.3, 4.8, 4.7,3.2, 9.2, 7.9, 7.7, 6.1, 4.9, and 4.0.

FIG. 5 shows use of template cross-correlation to distinguish spikes ofsimilar amplitude, but different shape. (A) Two portions of the rawtrace showing templates derived from spikes with different shapes. (B)Normalized cross-correlation plot showing the separability of thesedifferent units by different cross-correlations with the two templates.

FIG. 6 contains PST histograms of the eight units derived from the tenelement multi-array shown in FIG. 2B. In this case, no more than oneunit was derived from each channel, but, depending on the tip size andlocation in the retina, sometimes 2 or 3 distinct units are derivablefrom single channels using the template method. Case 9 shows theresponse of a unit to a left-to-right sweep of a bright bar, case 10 toa right-to-left sweep.

FIG. 7 is a drawing of an idealized cross-section of neural tissue(cross-hatched) showing only the somas of three neurons which are drawnto represent different layers of the neural tissue as in, for example,layers 1-6 of mammalian neo-cortex. The substrate of the prostheticdevice rests on the surface of the neural tissue, and individualelectrodes protrude at different distances from the substrate into theneural tissue. Four electrodes are represented, one is a stimulatingelectrode, and another is a receiving electrode. Two other electrodesillustrate a sharpened tip of an electrode, and the exposed end of theelectrode which protrudes beyond the substrate of the array.

FIG. 8. is a flow chart showing the elements of the feedback loop.

FIG. 9. is a flow chart showing the elements of the biosensing electrodecircuit.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Multielectrode Recording Array for Retina

As a recording device, the multi-electrode array system consists ofthree main elements: (1) the array and its mounting, (2) a datacollection system for the recordings, and (3) a data extraction,stimulus control and analysis system.

Described is a multielectrode array with metal or carbon fibers whichcan be used either to record various phenomena such as actionpotentials. If desired, the tips of carbon fibers from the substrate canbe coated with metals such as gold or silver to increase conductance ofelectricity. Also, if metal wires are used instead of carbon fibers, theexposed ends of these metal wires can be coated with a morebiocompatible substance such as carbon. Although what is described isthe embodiment for recording in the retina, the multielectrode recordingarray and system can be used to record from any neural tissue.

The retinal array and its mounting. The array consists of a set ofindividual microelectrodes mounted on a substrate that, in turn, isattached to a standard, high density 0.050 inch grid electricalconnector called a “header” or “interconnect” (Mill-Max, Oyster Bay,N.Y.) that is available in single and multiple row versions of variousnumber of pins. The substrate, which can be transparent (for example,mica or clear acetate) is glued to this header, and the electrodes arebonded to the substrate. The clear substrate has a roughly triangularshape, being wide at the connector end, and ending in a several hundredmicrometer wide tip region from whence the electrodes will emerge, asshown schematically in FIG. 1A. The individual electrodes are typically8 or 12 micrometer diameter carbon fibers (Thornel T-500, 12K, AmocoPerformance Products, Greenville, S.C.) insulated with polyurethane,although tungsten wire (1 mil, 25 micrometer diameter) has been used aswell.

The multielectrode array is made from a number of individual electrodes,and each electrode is suitable for recording, and can be tested forsuch. The carbon fibers typically are supplied in loose bundles. Lengthsof fibers are cut off the end of one of these bundles and the ends of anumber of the fibers are lightly pressed onto the edge of a piece ofdouble-sided (double-stick; Scotch) cellophane tape. The fibers are“fanned out” under a dissecting microscope so that some of theindividual fiber ends are separated. A three centimeter length of thinbare copper wire (#43AWG; 50 micrometers diameter) is advanced by amicromanipulator until the copper wire overlaps an individual carbonfiber by about 1 mm. A small drop of conductive bonding agent such ascolloidal silver paste (Ted Pella, Fadding, Calif.) is used to bond thecarbon fiber electrically and mechanically to the copper wire. Thewire-fiber assembly is then pulled off the tape, and the overlap regionis coated with a clear bonding agent such as polyurethane (DeltaCeramcoat Gloss Exterior/Interior Varnish, Whittier, Calif.) foradditional mechanical integrity and insulation.

The fibers are then electrolytically sharpened by bringing a highvoltage (1000-2000 volts DC) positively charged stainless steel point upto the carbon fiber as the negative pole, until a single small sparkerodes away a length at the tip of a few tens of micrometers. The carbonfiber is then insulated to within about 10 micrometers of the tip bylowering it into a small drop of polyurethane in a “U” shaped piecestainless steel wire, and advancing and retracting the fiber repeatedly(the tip itself never enters the polyurethane) until about 10-20 coatsof polyurethane are made from the tip region to the end at the overlapwith the copper wire, already insulated with polyurethane. This is doneunder a dissecting microscope to insure that the tip of the carbon fibernever enters the polyurethane drop. One of the principal reasons forchoosing polyurethane as an insulating material was its surface tensionand slow drying property made it possible to insulate the electrodes upto the tip in one step as above. The last step in making the individualmicroelectrode is to silver-plate the tip region by putting the tip in adrop of silver electroplating solution (Vigor Silver ElectroplatingSolution, B. Jadow & Sons, New York, N.Y. 10010) and passing a fewmicroamps of current (tip negative) for a few seconds under dissectingmicroscope observation. An individual electrode can be made in a fewminutes. The tip end of a polyurethane-insulated carbon fiber is shownin FIG. 2A.

The impedance of the electrodes can be determined during thesilver-plating procedure. Electrodes that have insufficient exposed tipdo not pass enough plating current and do not record well. This can becorrelated with the actual impedance. Because the plating can be donebefore the assembly, these can be rejected from use. The impedance ofthe electrodes in the array is measured with a constant current of 100nA at 1000 Hz. Most electrodes have impedances between 500 kΩ and 1 MΩ.A few electrodes have impedances between 1 and 5 MΩ and record lessreliably than those with lower impedances. Electrodes with impedanceshigher than 5 MΩs rarely yield usable recordings.

Instead of carbon fibers, wires composed of biocompatible metals such asplatinum, titanium, iridium, a platinum-iridium alloy, silver or othersimilar metals can be used. Use of these biocompatible wires wouldeliminate the need for a coupling between the carbon fibers and themetal wire embedded in the substrate.

In order for the array to be transparent for working in the retina (withonly the carbon fibers obstructing a small percentage of the lightpath), it is necessary to remove the copper wire portion of theelectrodes from the optical stimulus path. This is done by mounting theelectrodes on any transparent substrate (for example, mica or clearacetate) that can be advanced toward the retina at a 45 degree angle.Because retinal ganglion cells to be recorded lie in a single layer,this in turn means that the tips of the electrodes must be preciselyplaced so that when advancing along the axis of the electrodes, all tipslie in the same horizontal plane. The offset compensation for thisangular approach is shown in FIG. 1B.

The individual microelectrodes are placed in the array in layers.Although the numbers can be varied for any purpose, there are typically6 to 10 electrodes per layer. For the alignment, a small piece of nylonmesh (Monofilament Cloth, 20 microns, Small Parts, Miami Lakes, Fla.33014) should be attached temporarily at the tip of the substrate onwhich the electrodes will be bonded at the angle at which the retinalsurface will be encountered when the electrode array is advanced intoit. Just below (about 25 micrometers) this mesh is an acetate layer thatthe electrode tips will rest against during assembly. The individualmicroelectrodes are lowered by a micromanipulator so that the carbonfiber goes through the mesh, and rests against the clear acetate. Asmall drop of polyurethane bonds the carbon fiber in place near its exitfrom the substrate. When this first drop has set for a few seconds, asecond drop bonds the copper wire to the substrate, and the other end ofthe copper wire can then be released. Then the next microelectrode islaid in that layer, and the next, until the layer is finished. At thispoint, for additional layers, either a few coats of polyurethane areadded over the entire first layer, or a small piece of mica is bondedwith polyurethane to the top of the first layer, as the substrate forthe second layer. Finally, after all the individual microelectrodes areassembled, the copper wires are soldered to the pin connectors of theinterconnect-header.

Because carbon fibers are so flexible, the tips extend beyond thesubstrate less than about 1 mm, otherwise, surface tension in thesuperfusion fluid can cause the tips to bend towards each other and insome cases stick together. The big advantages of the millimeter gapbetween the electrode tips and the substrate, however, are that only thetips are in the retina, minimally disrupting it, and unimpeded flow ofsuperfusate over the tissue. In addition, in the retina, microelectrodesthat do not extend enough beyond the substrate tend only to record fromaxons, resulting in receptive fields that are not distributed tightlyaround the site of the array, as needed for examining population coding.FIG. 2B shows an array with two layers of electrodes whose centers are150-200 microns. The polyurethane coating on the 12 micrometer carbonfibers brings the total diameter of the insulated fiber to less than 20micrometers, so that inter-electrode spacing is less than 20 microns ifthey are packed together. Use of smaller diameter carbon fibers (e.g. 4microns) could produce electrodes whose spacing of centers would be lessthan 10 microns. Current array electrodes in the prior art are typicallyon much larger centers. Electrodes have some variability in theelectrode spacing. The electrode tips may be off-center by as much as±40 μm, and the tip extension by ±25 μm.

A retinal array of 16 elements can be constructed in about 12 hoursworking time at a station where all the supplies and jigs remain setup.The construction of each individual microelectrodes takes less than 10minutes, which includes bonding the carbon fiber to the copper wire,etching the carbon fiber to a point, coating the carbon fiber up to thetip with polyurethane, and then silver-plating the tip. The longest timeis used in adding each electrode to the assembly. The finishedmicroelectrode is lowered until the carbon fiber lies on the assembly,and then advanced until the carbon fiber tip passes through the correcthole in the mesh. This sometimes takes several attempts. The mostdifficult part is placing a very small drop of polyurethane on the fibernear the edge of the array to make the initial bond. Then a largerpolyurethane drop is applied farther up the fiber, and another over thecopper wire. This process can take 20-30 minutes per electrode. Thefinal process of soldering the free copper wire ends to the connectortakes only about 15 minutes, using a very small tipped batter solderingiron. Because the substrate for the array is glued to the side of theconnector, the contact points of the connector for soldering are off thesurface of the substrate and do not get contaminated by thepolyurethane. Neither does the low temperature soldering affect thesubstrate.

Mounting and manipulation of the retinal array embodiment. The array isplugged into a female connector that is attached to the array amplifier.This connector is mounted on a standard micromanipulator like that for asingle electrode, but with the following difference: the manipulator hasbeen equipped with rotation axes that allow the array to be rotated inpitch and yaw, in addition to the conventional axial movement. This isbecause, in the retina, the plane of microelectrode tips should be evenand parallel to the retinal surface to record simultaneously from asingle layer of ganglion cells. Slight rolls in the tissue requiretilting the array to achieve this, since the individual microelectrodesare not moveable relative to each other. Once properly oriented, thearray is used to explore different regions of the retina or other tissuein a manner similar to that used by a single electrode. Retinal ganglioncells, stained with Azure B (Amthor and Oyster, 1995) can be seenthrough the array, as the only portion of the field that is blocked isthe small percentage due to the carbon fibers in the field of theobjective lens (5×, Nikon), as shown in FIG. 2B. Stimuli in the systemfor use in the retina can be delivered through the array using theepi-illumination pathway via a 100% reflecting cube (XF125, OmegaOptical, Brattleboro, Vt.). For monitoring the stimulus location, a 50%reflecting “metallurgical” cube (Nikon) can be used to view the retina,array, and stimulus spot simultaneously.

Retinal recording and data acquisition embodiment. Although low noise,high input impedance amplifiers of up to four independent channels arerelatively commonly available (such as A-M Systems), units with 16 ormore channels are harder to find, and are much more expensive. Thepresent invention includes a simple, inexpensive amplifier system basedon the popular, high input impedance TL081 op amp that allows placementof a large number of channels in a small box at very low cost. Some ofits overall features contributed significantly to the efficiency andsuccess of the system which can be constructed with commonly availablematerials. For example, the TL081 operational amplifiers have beenavailable for more than 20 years, and now exist in dual and quadminiature surface mount versions, which allow considerable savings inspace and costs. The space saving allowed construction of small 4×4 inchboards with 16 complete channel amplifiers on each board, which in turnallowed mounting of all the electronics within a few inches of thearray.

Each amplifier consists of three stages: a preamp, an active bandpassfilter, and an output voltage limiter with optional gain and notchfiltering. The schematic of the amplifier is shown in FIG. 3. The preampis DC-coupled follower-with-gain, with a fixed gain of 10, and a highfrequency roll-off feedback capacitor. The DC coupling takes advantageof the high input impedance of the opamp. The preamp gain is limited to10 because the use of dissimilar electrode types and coating sometimesproduces offset junction potentials that can saturate the amplifiers athigher DC gains. The middle stage is a bandpass filter with a centerfrequency of about 2.5 kHz, as shown in the Bode plot in FIG. 3. Thelower limit of the bandpass filter limits the intrusion of 60 Hz linenoise, and other slow potentials such as due to movement of thesuperfusion fluid. The high limit excludes signals outside thoseproduced by neurons, and avoids aliasing the A/D converter.

The last stage is optional, and is primarily related to the particularA/D board used (i.e., Measurement Computing Corp., formerly known asComputer Boards) which operate on maximal ±10 volt input ranges. If thepower supply for the amplifier is greater than ±10 volts, then the A/Dinputs need to be protected. Additional gain can be added to boost thesignal further to “fill up” the A/D converter, which was more of anissue with a 12 bit than a current 16 bit A/D converter. The system alsocontains circuitry in some versions of this amplifier to further exclude60 Hz line noise, but careful control of electrode impedance and groundloops eliminated most of this noise without this portion of the circuit.Thus, proper choice of power supply and A/D converter can make thisstage unnecessary. If so, then, one can record with circuitry with onlytwo opamps, 5 resistors, and 4 capacitors per electrode channel.

Acquisition and Control of Retinal Data Embodiment. Although the arrayis manipulated much like single electrodes, recording with arraysdiffers from single electrode recording in one particularly significantaspect. With a single microelectrode, one changes the position and depthof the electrode until an adequate signal to noise ratio is obtained sothat spike times can be acquired with a threshold device such as aSchmitt trigger. But with an array, since one cannot move themicroelectrodes individually, there will be some proportion of thechannels that have poor signal to noise, or multiple spikes of similarheights. At least in experiments in rabbit retina, acquiring all thedata the array was capable of producing demanded analog data acquisitionand post-acquisition processing.

Data Acquisition. Adequate computer power and analog to digitalconversion cards have become very inexpensive recently: 64 channel 16bit A/D cards and the computers to house them are within the purchasingpower of most laboratories, although the expertise to program them maynot necessarily be. The system for generating impulses for testing andacquiring data runs on Microsoft Windows-based computers using 64channel (single ended) A/D boards from Measurement Computing Corp.Stimulus generation, all software for data acquisition, and analysis wasdeveloped using Microsoft Visual Studio 6.0 and the MeasurementComputing Universal Library. Direct programmatic access to videohardware, necessary for high quality graphical animations of thestimulus display, was accomplished using Microsoft DirectX 7.0.

Data acquisition and stimulus presentation were synchronized to thevertical retrace of the display monitor (100 frames/s). In recordings ofthe output of a photocell placed on the display monitor, the variabilityin the interval between the start of data acquisition and the time thatthe stimulus appeared at the photocell's position on the display was ina range of observed intervals ≦1 ms for 500 consecutive stimuluspresentations.

In the embodiment of the present invention for retinal recordings thereare 3 primary computational needs: (1) acquiring the analog data from 16or more electrodes at rates up to 10 kHz per channel, with minimaldelays for storing and processing these data, (2) coordinating this dataacquisition with the presentation of complex visual stimuli, which wasalso computer resource intensive, and (3) monitoring the performance ofthe array with a software-implemented “virtual oscilloscope” and givingfeedback to the experimenter about the cells spike responses, such aspost stimulus time histograms.

There are various ways of parceling out these three tasks, such as doingthem all on a single fast computer, or dividing the tasks among severalcomputers. One method presumes that the large demands of dataacquisition would tie up one computer completely, so that a secondcomputer, hand shaking with the first, would do stimulus generation andanalysis. Although this configuration works, it is not the correct onefor several reasons. The first is that newer A/D boards with fasterexpansion buses could acquire the analog data in bursts of a few secondsper stimulus without tying up the CPU very much, thereby leaving thehost CPU free for stimulus generation. The second reason is that theresource that has potentially an almost unlimited demand is the feedbackand analysis. If the machine doing this component has either of theother real time tasks (analog signal acquisition and stimulusgeneration), then there is a very finite limit to what processing can bedone on the data acquired from a particular stimulus, without creatingan undue delay before the next can be generated.

This leads to another configuration. One machine does both dataacquisition and stimulus generation. This is a dual monitor machine withone monitor displaying stimuli to be projected onto the retina, and asecond monitor displaying control information to the experimenter.Difficult hand-shaking tasking is eliminated because all real timefunctions are done on one machine, an ordinary off-the-shelf Pentium.The second computer, called Data Spy, does the virtual oscilloscopes foreach channel, and all the analysis and cell response display functionsasynchronously, by accessing files written by the real-time machine overthe local area network (LAN). Since this machine functionsasynchronously by accessing files on its hard drive, different types ofanalysis can be selected at different times, even during a data run. Ifdata arrives faster than it can process, it simply falls behind a fewstimuli, but this has no effect on data collection. It even allows theoperator to stop a given kind of analysis, and start a different onefrom scratch, from the first stimulus, in the middle of the data run.

Software for using the array consists of 3 main components: data(voltage) acquisition, stimulus generation synchronized with dataacquisition, and output storage and user display/interface.

Array outputs are electrically connected via integral connector to theamplifier described above. Output of this amplifier is electricallyconnected to a storage device, such as analog to digital converter boardin a Windows computer. Software contained in the system controls thestate and operation of the analog to digital converter board, andsynchronizes acquisition with stimulus generation (B) functions, anduser display/output (C) functions. The entire system can operate on asingle computer, or multiple computers. Software can control propertiesof the amplifier circuitry described above, including application ofvoltages or pulse trains to specific array elements under softwareprogram control.

Software synchronized acquisition of array from the data withpresentation of graphic pictures on a windows computer monitor. Softwarecan generate sounds, or voltage outputs to control devices such as pumpsand relays, or other instrumentation.

In the present embodiment, stimulus synchronization with dataacquisition uses “Direct-X” Microsoft software, but could also use other“low-level” control software. Stimuli generated include full screengraphics, changeable at every frame, at frame rates of 100 Hz or higher,with data acquisition synchronized to a specific phase of the verticalretrace pulse of the computer monitor. This allows synchronization ofthe data acquisition to be within one sample time (<1 millisecond at4000 Hz sampling rate, for example) of a specific phase of the monitorvertical retrace, and therefore of the stimulus presentation.

Stimulus presentation described above include both the presentation ofpre-computed image files, and the movement and merger of images bydynamic computation of graphic images at the monitor frame rate of 100Hz or better. Examples of images synchronized with data acquisitioninclude moving rectangular objects, moving gratings, and movement of anyarbitrary bitmap, and appearance and disappearance of these objects atuser specified times during the experiment.

Operation of data acquisition synchronized with stimulus generationproduces output files that are initially stored in the computer RAMmemory, and also, at intervals, stored on the computer hard drive, ortransmitted or a network connection to another computer or other device.Output files can contain entries that consist of the voltage/currentderived from or applied to any or all of the array elements, the exactanalog to digital sample number or time, the status or the stimulusdisplay, that status of any output device such as a pump or relay orother instrument controlled by the software. The files may containheader or other experimental information, and may contain user entriesmade before or during the data acquisition, and any results ofprocessing the data acquired in the same or other files.

The software also generates a user interface that reports the result ofdata acquired from each, any or all of the array elements, the status ofthe elements, summary data that combines information from multipleelements, instructions to the user, graphical plots of the dataacquired, comparison of the data acquired with other, previouslyacquired data or with mathematical models, suitable user interface forcontrol of the array elements in voltage or current acquisition mode, oroutput mode, suitable user interface for control of the graphicaldisplay or stimulus or interface with other connected devices, andcontrol of network interchange of information. Such user interface canbe on the computer monitors or via audio output.

Spike extraction. At the end of an experiment, there are a set of largefiles, each of which contains the analog data recorded at 4 to 10 kHzper channel, for every channel of that array and for every stimulus inthat data run. Next a file is generated that contains the times ofoccurrence of the spikes from each cell recorded, for each stimulus.Storing the analog “raw” data and then extracting spike times requiressome processing, but has a number of important advantages. First, theprecision of the timing of the spikes is much better when they arelocated in a 4 kHz analog trace, than reading a clock when the spikewaveforms cross the threshold of a hardware Schmitt trigger set for theentire data run, because the actual peak of the waveform or otherattribute can be located in time precisely. Second, some of theelectrodes in the array are necessarily not optimally positioned toyield the largest signal to noise spikes, or have spikes from severalcells, so that the ability to detect and discriminate spikes byprocessing the analog signal offline is vastly superior to the one shotSchmitt trigger hardware method.

An outline of the extraction method follows, which is similar to thatused by Nordhausen (1996). For each channel, the first 5-10 stimuli aresearched for candidate spikes, based primarily on spike amplitude. Iftrue spikes appear to be present, even if of low signal to noise, atemplate building mode is entered. In this mode, the record is searchedfor more of these likely spike events, and they are individuallyselected to form an average template. Once this template is stable, alarge portion of the data run for that channel is cross correlated withthe template. The distribution of normalized cross correlation values isplotted, as well as the peri-stimulus time (PST) histograms as afunction of the cross correlation value. True spikes will tend tocluster around a single peak in the distribution of correlation values,and will also tend to fall in the same PST bins. The PST histograms canbe seen to broaden, or shift, when noise spikes, or spikes from adifferent cell of lower cross correlation value are included.

Once the first set of spikes are identified from a record, they areremoved and the record is processed again to produce a new template.This iterative process allows the extraction of up to four differentspikes from a given record. This process is repeated for each record,and a new file is created which has spike times from each cell for eachchannel, using the actual channel each cell was recorded for the firstspike type on each channel, and pseudo-channels for multiple spikesextracted from each channel. From these files are generated typicalplots such as the PST histograms, cross correlation histograms, polarplots for movement, and so forth.

FIG. 4 shows the 10 “raw” analog data-captured traces from a 10 elementarray. Even at this low resolution scale, it is clear that 5-6 of theelectrodes have easily discernable spikes; the spikes on some channelsare small and not very evident at this scale. In the retina, virtuallyall the electrodes in these arrays work in that they record spikes atsome retinal location or depth. At any particular depth and placement,about half of the elements will record easily discernable spikes at onetime, with some electrodes recording several separable units. Thus, thetotal number of recordings recovered is typically on the order of thenumber of electrodes in the array. As expected, larger electrode tipsplaced in areas of the retina of high ganglion cell density, such as thevisual streak, record more cells per electrode, while smaller tips insparser regions record fewer.

FIG. 5 shows the use of the template method for reliably extractingmultiple spikes from a single microelectrode recording. The greatadvantage of templates is the ability to separate different spikespurely on the basis of shape, when they could not be reliablydistinguished on the basis of pure amplitude. All channels are processedoffline in this manner, although the system contains a hardware Schmitttrigger as typical in single microelectrode recording, and a softwarethreshold discrimination on the virtual oscilloscopes to get real timefeedback about the type of cells currently being sampled and thestability of the recordings.

FIG. 6 shows the peri-stimulus time (PST) histograms of 8 unitsrecovered from the traces in FIG. 4, which in turn were obtained fromthe 10 electrode array in FIG. 2B. Eight units were recovered. Eachrecovered unit appears to be from a distinct cell from the appearance ofthe PST histograms, although we did not directly examine the waveformsacross channels. Firing cross correlation functions can be computed inwhich a suspiciously large peak at a single fixed delay would triggersuch an examination.

Aspects of the procedure (beyond those considerations common to singleelectrode recording) that were essential for success were primarilyusing electrodes with fairly long tips that extended at least a few tensof micrometers beyond the substrate, mounting accuracy, and the abilityto tilt the array in pitch and yaw to get all the tips in the samehorizontal plane to match that of the tissue to be recorded. A number ofconfigurations were tried with short tips emerging less than 10micrometers from the substrate. In retina these resulted primarily inaxon recordings (ganglion cell axons form a layer proximal to theganglion cell soma layer). In retina such recordings are not preferredbecause the receptive fields of axon recordings are not confined to theregion near the electrode tip, but may be found considerable distancesaway, and the overlap among such receptive fields was minimal.

Getting the electrode tips all in the ganglion cell layer is alsocritical in retinal recordings. This involved accuracy in both mountingand placement. Probably the most difficult part of the assemblyprocedure is the mounting. Although inter-electrode spacings of 20micrometers or less can be achieved routinely, controlling the exactdepth of all the tips with manually applied drops of the polyurethanebonding agent requires skill and patience, and the spacing is notentirely uniform in either the X-Y, or Z dimensions (as can be seen inFIG. 2B). Nevertheless, even perfect electrode-electrode depthalignments proved to be useless without the ability to align the arraywith the ganglion cell layer. Prior to installing the 2 tilt axes, manyearly arrays, some as large as 32 elements, appeared only to have a fewviable channels and were needlessly discarded.

The instant invention has definite advantages compared to conventionalsingle cell recoding. One is stability. Since the array has adistributed, but punctuated, “footprint” across the retina, there islittle movement over time of the tissue with respect to the array overtime. Recordings with this array are typically stable for 4-6 hours, andusually degrade gracefully even after that period. The arrays are alsovery robust. To generate the photomicrograph in FIG. 2B, we actuallydrove the array tips onto the surface of the glass graticule to get thetips and graticule in focus simultaneously, without damaging the array.Arrays can be repeatedly driven into the bottom of a plastic Petri dish,with no apparent damage because the carbon fiber tips flex, and returnto their original position without breaking or taking on a permanentbend. So far, very few electrode elements have “dropped out” of functionin any of our arrays over many months of use.

The array allows recording mammalian cells with a multi-electrode arraysystem whose construction is within the reach of virtually anyelectrophysiology laboratory. The array is robust, and holds stablerecordings for many hours, and is usable for months or longer. The neartransparency of the array allows visualization of the tissue through it.This is particularly useful when recording from retina, because visualstimuli can reach the retina through the array. Transparency is alsoimportant in recording from brain slices or tissue cultures, and alsohas the advantage of allows lab personnel to view the underlying tissuewithout removing the array. The instant invention is also useful forcombining array recording with optical imaging. In addition, because therecording elements of the arrays are carbon fibers, they are potentiallyuseful for recordings other than voltage, by the use of coatings thatrespond to pH or the presence of any organic chemical.

The fact that these array recordings are achieved with rather mundaneelectronic amplification indicates that the signal-to-noise achievedwith the array elements is comparable to single electrode recordingswith standard microelectrodes. The individual microelectrodes in thesearrays can be made in less than 10 minutes. A layer of 8-10 electrodescan be assembled in a few hours. Although most of the arrays we haveused to date have had 16 or fewer elements, the only limit to thisnumber is the patience of the array builder. Inter-element spacings ofless than 20 micrometers are easily achieved.

The closest extant array to the one reported herein is that reported byKruger and Bach (1981), which assembled individual microelectrodes intoan array with a spacing of 160 micrometers, a spacing much larger thanthat allowed by the present invention (less than 20 micrometers).However, the array of Kruger and Bach, designed for recording in cortex,was not transparent, and thus not suitable for the retinal recordingconfiguration we have in mind, such as stimulating through the electrodearray. Kruger and Bach also used metal wire whose tips extended 2.5 mmfrom the substrate, a larger distance than in the present inventionwhich uses carbon fibers. Moreover, the manufacture of our individualmicroelectrodes is far easier and more controllable than that in manyother arrays, and the use of carbon fibers has potential applicationsfor electrochemical detection (which we have not explored).

One particular advantage of achieving electrode spacing less than 20micrometers is the ability to use rather small tips to over-sample anarea of the tissue so that a high proportion of the cells are recorded,but on separate channels. This is, in turn, related to the problem ofrecovering multiple cells from individual channels when it is desired todo firing cross correlation measurements. When multiple cells are pickedup on a single channel, spikes that occur at approximately the same timewill necessarily have overlapping waveforms. The resultant waveform willcorrespond to neither of the templates of the two cells, and, in theworst case, may actually have no amplitude component above the thresholdset for considering events as spikes. Because cross correlation studies(Singer et al., 1997) have most frequently shown peaks of firingcoincidence near zero milliseconds delay, the problem of spike collisionin multiple cell per electrode recordings is worst at the very delay ofmost interest. Therefore, it is clearly desirable to use more electrodesat finer spacing than the alternative.

II. Prosthetic Device

Carbon fibers are intrinsically a suitable material as electron donorand acceptors, and they therefore can be used without any coating inboth receiving voltage signals from neurons and stimulating neurons withvoltage signals. They can also have coatings such as used for recordingto enhance either receiving or stimulating. Stimulation and receivingcan be combined in the same array. Some array elements may receivevoltage signals, and these recordings could be filtered by a band-passamplifier, which would then either then other array elements would usethe output of that processing to stimulate neurons to control, enhance,replace CNS function, either lost due to disease or degeneration, or toenhance normal function such as in a direct CNS interface.

A small portion of a prosthetic device for stimulating and receiving inneural tissue with only minimal invasiveness is shown in FIG. 7. Theprosthetic device described herein can be used for stimulation atmillivoltage levels, as well as for receiving millivoltage signals, andcan be implanted as a prosthetic device in a living body. Additionally,another embodiment of the same invention may have electrodes 1 which arefor stimulating neurons, and may have other electrodes 2 for receivingvoltage signals from the neural tissue, and the voltage signals receivedmay be used to alter the activation pattern of the stimulatingelectrodes in the same array. The sharpened end 3 of the electrode isthe most distal portion which is revealed by discontinuation of theelectrical insulator. The exposed end 4 of the electrode is that portionof the electrode which is not housed within the substrate 5. Thecouplings of the carbon fibers and metal wires are 6. The electricalinsulator covering a portion of each electrode is 7. The configurationof the array when used as an implanted prosthesis is different than isshown in FIG. 1A, 1B, and 2B. The structure of the array for use as aprosthetic device (i.e., for stimulating or recording in neural tissue,or for doing both simultaneously through electrodes in the samemultielectrode device) would be different than the structure of thearray used for recording in the retina. One of the difficulties ofstimulating neural tissue artificially in a manner that would replicatenatural stimulation is: how does one deliver enough stimuli to tissuethat is multi-layered or otherwise too thick to allow stimulation onlyon the surface of the structure, without also damaging the underlyingtissue by driving electrodes into the tissue and damaging it thereby?Another problem is that metal electrodes present the possibility ofcorrosion, especially when implanted for long periods of time. A numberof efforts have been made to create such devices, but the presentinvention solves these and other problems much better than any existingdevice.

Spacing of individual electrodes in the array can be less than 10micrometers between centers. The electrode spacing is a function of thewidth of the carbon fibers; i.e., the wider the fibers the more spacingrequired. The insulation layer (e.g., polyurethane) on each electrode isless than 2 microns. Thus, five micron carbon fiber electrodes, forexample, could be on centers less than 10 microns apart because the onlyadditional width required between electrodes is the insulation for eachelectrode. As a further example, a 12 micron carbon fiber array couldhave electrodes whose centers are spaced apart less than 20 microns.Choice of the width of fibers depends on the desired length of extensionbeyond the substrate (herein referred to as “protrusion distance”). Awider carbon fiber is used when greater protrusion distance is required.

The software described above can control, by prior instruction sets,manipulation of the array within or between samples in the recordingdevice and, in the prosthetic device, placement of voltages or currentson individual array elements in conjunction with data obtained up to thepresent instant. For example, for a neural prosthesis used to stimulateneural tissue such as cortex or the spine, the software could acquireneural data from any or all array elements, process the data accordingto a mathematical model, and output pulses on any or all array elementsto stimulate neurons in the vicinity of the array, or in some otherarray, or control some other device. This is an advantageous feature ofthe array: it allows simultaneous receipt of voltage signals fromneurons and stimulation of neurons with voltage signals. This hasimportant advantages for applications in the nervous system in whichfeedback loops perform vitally important neural processing ofinformation and stimulus. For example, it is well known that most axonsin the LGN of the thalamus project to primary visual cortex(thalamo-cortical projections), also known as area V1. It is also knownthat the largest output of axons from visual cortex V1 goes to thethalamus where certain vision-related processing occurs. One theory isthat these cortico-thalamic projections are important in determiningattention, as salient features in cortex are somehow excited further andweaker features are inhibited in the thalamus. The unprecedented abilityof the present invention to pack large numbers of electrodes into a verysmall area means that the receiving electrodes could serve a functionsimilar to the cortico-thalamic feedback projections by connecting thereceiving electrodes to a band-pass amplifier or other devices implantednearby whose output would then, through coupling with the stimulatingelectrodes, inhibit the weaker features in the visual field butstrengthen or excite the more salient. These stimulating electrodeswould be analogous to thalamo-cortical projections. Thus, simultaneousstimulation and recording could allow development of visual attention,an unprecedented feature for artificial vision. This is only one exampleof how feedback loops could be important for neural prosthetics. FIG. 8is a diagram of a feedback loop which could be constructed to utilize anartificial feedback mimicking that of the CNS and PNS. FIG. 8 contains areceiving electrode 2, extending through the array's substrate 5,coupled to a pre-amplifier 8, then coupled to a band-pass amplifier 9,then coupled to an output controller 10, then coupled to a currentgenerator 11. A current source 12 is also coupled to the currentgenerator 11.

Further, the ability to convert a stimulating electrode to an electrodefor receiving voltage signals within the same multielectrode array wouldallow flexibility for adjusting the pattern of activation of a givenarea of neural tissue with feedback and direction from a human subject.This type of feedback, analogous to that of an epilepsy patient underlocal anesthetic during surgery to remove the affected cortical tissue,would allow a doctor to make adjustments in the invention's stimulationpattern with instructions based upon the patient's sensory perceptions.Additionally, adjustments could be made after conclusion of surgery.

Another significant aspect of the present invention is that theindividual electrodes can be varied in length to target different layersor areas of neural structures so as to ensure stimulation and recordingin areas much more diverse than previous devices have allowed. Thisvariable protrusion distance on different electrodes in the sameprosthetic device is a major advance over the prior art. For instance,neo-cortex in most mammals is 2-4 millimeters thick and has identifiablelayers, and stimulating and recording in different layers would bepossible with the current invention by varying the lengths of themultiple protrusion distances of the individual electrodes. Thesubstrate from which the individual electrodes protrude would restagainst the surface of the neural structure and the individualelectrodes would pierce the surface to the desired depths. Becauseindividual electrodes can be less than 10 microns apart (depending onthe thickness of the carbon fibers), these electrodes can slide throughthe tissue to deeper layers or thicknesses with very minimal damage tothe neurons in the upper layers. This aspect of the invention isdepicted in FIG. 7.

Individual electrodes of different protrusion distances are insulated toa position in the neighborhood of the etched point (as described above)so that the sharpened point is the only portion of the protrusiondistance un-insulated. This allows delivery of charge to a preciselocation. For example, assuming a protrusion distance of 2 mm, thecurrent would travel only to tissue at the exposed sharpened andun-insulated tip which is less than 15 microns.

For electrophysiology, the software can specifically display the outputof each, any, or all array elements as a result of the stimulus to forma “map” of the response versus stimulus parameter such as position,intensity or other parameter. The software can display post stimulustime histograms of the firing of action potentials of neurons recordedby the array. The software can display, following analysis by thesoftware, of cross channel data features such as synchronous firing ofneurons, or detection of particular firing patterns in either single ormultiple channels. The software can allow interactive setting ofthresholds or other parameters or online analysis of data acquired, anddisplay during the data acquisition, results from all data acquiredprior to the present instant.

III. Bio-Sensing Device

The carbon fiber array may also be used as a sensor for organiccompounds, and this sensing can be done separately or simultaneously (inthe same array) with electrophysiological recording or stimulation. Thearray can be implanted or placed temporarily in vivo with coatings orreleasable substances which enhance tissue compatibility or neuralinterface, such as neural growth factors, cell adhesion molecules, oreven stem cells.

Carbon fibers act as electron donor or acceptors and so can participatein reduction—oxidation detection of oxidizable or reducible biologicalsubstances such as neurotransmitters like dopamine, by application of avoltage to the fiber, and monitoring of current flow in the presence ofthe biological substrate. Coatings can be applied to the fiber tips formore specific detection. The array is mounted on a standard connector,and therefore can be disposable. Multiple sensing elements can be placedin a very small space, such as a single droplet of fluid, capillary bed,or any biological substance. Multiple voltages can be applied todifferent array elements, either simultaneously, or sequentially, toenhance the ability to detect and identify a particular biochemicalspecies. Different coatings can be used on different array elementseither to (1) enhance the specificity of detection and identification ofa chemical species, or (2) allow simultaneous detection of multiplebiochemical or chemical substances. For example, by means of anamperometry device, the array could make physiological measurementsrelevant to blood pressure (any combination of neural activity andbio-sensing) and produce an output to control other neurons affectingblood pressure, or activate a pump that released a substance thataffected blood pressure. Amperometry involves applying a fixed orpulsatile voltage between 2 electrodes, and determining the currentpassing in the circuit between the electrodes. Depending on theelectrodes, the current is related to the concentration of chemicalspecies that are detectable by the electrodes at the voltage potential.Amperometry devices are produced, for example, by Abtech Scientific,Inc. of Richmond, Va.

FIG. 9 is a flow chart showing the circuit for the biosensing electrode,with a receiving electrode 2 extending beyond the substrate to acoupling 6 with a metal wire 16, which is coupled to an amperometrydevice 14, which is coupled to a voltage source, which is coupled to areference wire 13, which also extends beyond the substrate 5 and intothe biological material being sampled.

REFERENCES

Amthor F R and Oyster C W. Spatial organization of retinal informationabout movement detection. Proc. Natl. Acad. Sci. USA., 1995; 92(9):4002-4005.

Amthor, F R, Tootle, J S, Yildirim, Abidin, A new transparent multi-unitrecording array system fabricated by in-house laboratory technology. J.Neurosci. Methods, 2003; 126(2): 209-219

Grumet A E, Wyatt, Jr. J L and Rizzo, III J F Multi-electrodestimulation and recording in the isolated retina. J. Neurosci. Methods,2000; 101: 31-42.

Heuschkel M O, Fejtl M, Raggenbass M, Bertrand D and Renaud P. A threedimensional multi-electrode array for multi-site stimulation andrecording in brain slices. J. Neurosci. Methods, 2002; 114: 135-148.

Meister M, Pine J and Baylor D A. Multi-neuronal signals from theretina: acquisition and analysis. J. Neurosci. Methods, 1994; 51:95-106.

Nordhausen, C. T., Maynard, E. M., and Normann, R. A. Single UnitRecording Capabilities of a 100 Microelectrode Array. Brain Res., 1996;726:129-140.

Sandison M, Curtis A S G and Wilkinson C D W. Effective extra-cellularrecording from vertebrate neurons in culture using a new type ofmicro-electrode array., J. Neurosci. Methods, 2002; 114: 63-71.

Singer W, Engel A K, Kreiter A K, Monk, M H J, Neuenschwander S andRoelfsema P. Neuronal assemblies: necessity, signature anddetectability. TINS, 1997; 1(7):252-261.

Thorpe S, Delorme A and Van Rullen, R. Spike-based strategies for rapidprocessing. Neural Networks, 2001; 14:715-725.

1. A multielectrode array for receiving voltage signals from neurons,the multielectrode array comprising: A substrate; At least twoelectrodes partially contained in said substrate, each of said at leasttwo electrodes having a carbon fiber with an exposed end extendingbeyond the substrate, an unexposed end within the substrate, and acenter, said exposed end capable of being embedded in neural tissue, andAn electrical insulator covering at least a portion of each of the atleast two electrodes.
 2. The multielectrode array as in claim 1, whereeach of said at least two carbon fibers has any combination of at leastone of the characteristics independently selected from the groupconsisting of: a coating on the exposed end, a sharpened tip, and variedlength of the exposed end. 3-10. (canceled)
 11. A multielectrode arrayfor receiving voltage signals from neurons, the multielectrode arraycomprising: A substrate; At least two electrodes partially contained insaid substrate, each of said at least two electrodes having abiocompatible wire with an exposed end extending beyond the substrate,an unexposed end within the substrate, and a center, said exposed endcapable of being embedded in neural tissue, and said at least twoelectrodes being spaced from one another so that the spacing between thecenters of said exposed ends of each said biocompatible wire does notexceed 45 microns; and An electrical insulator covering at least aportion of each of the at least two electrodes. 12-24. (canceled)
 25. Amultielectrode array for receiving voltage from neurons and stimulatingneurons with voltage signals, the array comprising: A substrate; Atleast one receiving electrode partially contained in said substrate,each said at least one receiving electrode having a carbon fiber with anexposed end extending beyond the substrate, an unexposed end within thesubstrate, and a center, said exposed end capable of being embedded inneural tissue, said carbon fiber's unexposed end coupled to a metalwire's first end, said metal wire's second end being coupled to theinput to a pre-amplifier; At least one stimulating electrode partiallycontained in said substrate, each said at least one stimulatingelectrode having a carbon fiber with an exposed end extending beyond thesubstrate, an unexposed end within the substrate, and a center, saidexposed end capable of being embedded in neural tissue, said carbonfiber's unexposed end coupled to a metal wire's first end, said metalwire's second end being coupled to the input to a current generator; andAn electrical insulator covering at least a portion of the said at leastone receiving electrode and the said at least one stimulating electrode.26. The multielectrode array as in claim 25, where each of said at leasttwo carbon fibers has any combination of at least one of thecharacteristics independently selected from the group consisting of: acoating on the exposed end, a sharpened tip, and varied length of theexposed end. 27-30. (canceled)
 31. A multielectrode array forstimulating neurons with voltage signals and for receiving voltagesignals from neurons, the multielectrode array comprising: A substrate;At least one stimulating electrode partially contained in saidsubstrate, each at least one stimulating electrode having abiocompatible wire with an exposed end extending beyond the substrate,an unexposed end within the substrate, and a center, said exposed endcapable of being embedded in neural tissue and said unexposed endcoupled to the input to a current generator; At least one receivingelectrode partially contained in said substrate, each at least onereceiving electrode having a biocompatible wire with an exposed endextending beyond the substrate, an unexposed end within the substrate,and a center, said exposed end capable of being embedded in neuraltissue and said unexposed end coupled to the input to a pre-amplifier;and An electrical insulator covering at least a portion of the said atleast one receiving electrode and the said at least one stimulatingelectrode.
 32. The multielectrode array as in claim 31, where each ofsaid biocompatible wires has any combination of at least one of thecharacteristics independently selected from the group consisting of: acoating on the exposed end, a sharpened tip, and varied length of theexposed end. 33-36. (canceled)
 37. A multielectrode array for receivingvoltage from neurons, stimulating neurons with voltage signals, and forproviding feed-back between neurons, the array comprising: Saidsubstrate; At least one receiving electrode partially contained in saidsubstrate, each said at least one receiving electrode having a carbonfiber with an exposed end extending beyond the substrate, an unexposedend within the substrate, and a center, said exposed end capable ofbeing embedded in neural tissue, said carbon fiber's unexposed endcoupled to a metal wire's first end, said metal wire's second end beingcoupled to the input to a pre-amplifier; At least one stimulatingelectrode partially contained in said substrate, each said at least onestimulating electrode having a carbon fiber with an exposed endextending beyond the substrate, an unexposed end within the substrate,and a center, said exposed end capable of being embedded in neuraltissue, said carbon fiber's unexposed end coupled to a metal wire'sfirst end, said metal wire's second end being coupled to the input to acurrent generator; At least one feedback loop constituting a systempartially contained in said substrate, containing a first carbon fiberwith an exposed end capable of being embedded in neural-tissue, saidfirst carbon fiber's unexposed end coupled to a first metal wire's firstend, said first metal wire's second end coupled to the input to apre-amplifier, the output of said pre-amplifier coupled to the input toa band-pass amplifier, the output from said band-pass amplifier coupledto the input to an output limiter, the output of said output limitercoupled to a current generator, said current generator coupled to thesaid second end of a second metal wire and also to a current supply, thesaid first end of said second metal wire coupled to the unexposed end ofa second carbon fiber; the said exposed end of said second carbon fibercapable of being embedded in neural tissue; and Said electricalinsulator for each of the said at least one receiving electrode, said atleast one stimulating electrode, and said at least one feedback loop.38. The multielectrode array as in claim 37, where each of said at leasttwo carbon fibers has any combination of at least one of thecharacteristics independently selected from the group consisting of: acoating on the exposed end, a sharpened tip, and varied length of theexposed end.
 39. The multielectrode array as in claim 37, where theoutput limiter is deleted. 40-45. (canceled)
 46. A multielectrode arrayfor stimulating neurons with voltage signals and for receiving voltagesignals from neurons, and for providing feed-back between neurons, themultielectrode array comprising: A substrate; At least one stimulatingelectrode partially contained in said substrate, each at least onestimulating electrode having a biocompatible wire with an exposed endextending beyond the substrate, an unexposed end within the substrate,and a center, said exposed end capable of being embedded in neuraltissue and said unexposed end coupled to the input to a currentgenerator; At least one receiving electrode partially contained in saidsubstrate, each at least one receiving electrode having a biocompatiblewire with an exposed end extending beyond the substrate, an unexposedend within the substrate, and a center, said exposed end capable ofbeing embedded in neural tissue and said unexposed end coupled to theinput to a pre-amplifier; At least one feedback loop constituting asystem partially contained in said substrate, containing a firstbiocompatible wire with an exposed end extending beyond the substrate,an unexposed end within the substrate, and a center, said firstbiocompatible wire's unexposed end coupled to the input to apre-amplifier, the output of said pre-amplifier coupled to the input toa band-pass amplifier, the output from said band-pass amplifier coupledto the input to an output limiter, the output of said output limitercoupled to a current generator, said current generator coupled to thesecond end of a second biocompatible wire and also to a current supply,the said first end of said second biocompatible wire capable of beingembedded in neural tissue; and Said electrical insulator for each of thesaid at least one receiving electrode, said at least one stimulatingelectrode, and said at least one feedback loop; and An electricalinsulator covering at least a portion of the said at least one receivingelectrode and the said at least one stimulating electrode.
 47. Themultielectrode array as in claim 46, where each of said biocompatiblewires has any combination of at least one of the characteristicsindependently selected from the group consisting of: a coating on theexposed end, a sharpened tip, and varied length of the exposed end. 48.The multielectrode array as in claim 46, where the output limiter isdeleted. 49-54. (canceled)
 55. A biosensing device, comprising: Asubstrate; At least two biosensing electrode circuits, each said atleast two biosensing electrode circuit containing a biosensing electrodehaving a carbon fiber with an exposed end extending beyond thesubstrate, an exposed end within the substrate, and a center, saidcarbon fiber being coupled with the first end of a metal wire, saidmetal wire's second end-being coupled with an amperometry device, saidamperometry device being coupled with a voltage source and apre-amplifier, said voltage source being coupled with a reference wire;and An electrical insulator covering at least a portion of each of thebiosensing electrodes and the reference wire.
 56. The biosensing deviceas in claim 55, where each of said at least two carbon fibers has anycombination of at least one of the characteristics independentlyselected from the group consisting of: a coating on the exposed end, asharpened tip, and varied length of the exposed end. 57-60. (canceled)61. A biosensing device, comprising: A substrate; At least twobiosensing electrode circuits, each of said at least two biosensingelectrode circuits containing a biosensing electrode having abiocompatible wire with an exposed end extending beyond the substrate,an exposed end within the substrate, and a center, said biocompatiblewire being coupled with an amperometry device, said amperometry devicebeing coupled with a voltage source and a pre-amplifier, said voltagesource being coupled with a reference wire, and said at least twobiosensing electrodes being spaced from one another so that the spacingbetween the centers of said exposed ends of each said carbon fiber doesnot exceed 20 microns; and An electrical insulator covering at least aportion of each of the biosensing electrodes and the reference wire. 62.The biosensing device as in claim 61, where said biocompatible wire hasany combination of at least one of the characteristics independentlyselected from the group consisting of: a coating on the exposed end, asharpened tip, and varied length of the exposed end. 63-65. (canceled)66. The multielectrode array as in claim 1, wherein the at least twoelectrodes are selected from the following: receiving electrodes andstimulating electrodes.
 67. The multielectrode array as in claim 1,wherein said at least two electrodes being spaced from one another sothat the spacing between the centers of said exposed ends of each saidcarbon fiber does not exceed 20 microns.
 68. The multielectrode array asin claim 1, wherein said at least two receiving electrodes being spacedfrom one another so that the spacing between the centers of said exposedends of each said carbon fiber does not exceed 45 microns.